Many species of microorganisms, particularly bacteria and unicellular fungi such as yeasts, can be identified nowadays quickly and with low error rates by means of mass spectrometry. The identification is routinely done by computing similarity values between a mass spectrum of the disrupted (lysed) microbes, particularly their soluble proteins, and similar types of reference mass spectra of known microorganisms. If the similarity values exceed certain limit values, family, genus, species and even strain can be identified. This very fast and low-cost method of identifying microorganisms has proven to be extraordinarily successful, both in large scale studies and in the daily routine in many microbiological laboratories. Depending on the instrument, 48 to 384 microbial samples can be determined at the same time; identification takes only minutes from the end of culturing a colony to the identification. The method has very low error rates, much lower than the error rates of conventional microbiological identification methods, and lower even than those of DNA analyses. There are meanwhile mass spectrometers, associated evaluation programs and libraries of reference spectra on the market which are certified as IVD products for medical diagnostics in accordance with the German Medical Devices Act (MPG) and other, national or international regulations and guidelines.
As is usual in general parlance, the term “antibiotic” means a pharmacologically active substance for the treatment of microbial infectious diseases and also substances for disinfection. The successes of penicillin led to the search for and discovery of many other antibiotics. There are broad-spectrum antibiotics, which are effective against many families of microbes, and narrow-spectrum antibiotics, which are specifically effective against individual microbe species.
Ever since penicillin was used as the first pharmacologically active substance, microbial strains have increasingly developed various types of resistance to different types of antibiotics, or acquired them from other microbes, i.e. they have acquired characteristics which allow them to weaken the effect of antibiotic substances or to neutralize their effect completely. Resistances are unfortunately common meanwhile; microbes occurring in hospitals are today resistant in the main. In some cases, it is possible to predict the resistance of a microbe transmitted within the hospital to the antibiotics usually used in the hospital; this does not apply to infections which were acquired outside the hospital.
The success of a therapy for microbial infections, which are usually life-threatening in acute situations such as sepsis, or as a secondary infection during an existing primary illness (or primary infection), often depends on the first administration of an antibiotic being effective. Targeted administration requires not only that the pathogen is identified as quickly and correctly as possible, but also that its resistance to different antibiotics is determined as quickly as possible. The conventional determination of resistance consists of a culture in the presence of an antibiotic, but this unfortunately takes a very long time: 24 to 48 hours.
In addition to culturing in the presence of antibiotics, there are also genetic methods of determining resistances. There, a resistance is detected by detecting known resistance genes in the genome of the pathogen in question. An advantage of the genetic methods consists in the fact that the resistance genes can be amplified by techniques such as polymerase chain reaction (PCR), and thus the time needed for the analysis is no longer determined by the growth rate of the bacteria. The disadvantages are that they are more expensive than routine methods and are not functional tests. A resistance gene may be present, but not be expressed, which means the bacterial strain under investigation is not resistant, but the method detects it as being resistant.
The patent DE 10 2006 021 493 B4 (V. M. Govorun and J. Franzen, 2006, corresponding to GB 2 438 066 B and U.S. Pat. No. 8,293,496 B2; called “Govorun” in the following) discloses mass-spectrometric methods for the resistance determination of microbes.
In one embodiment, protein profiles of the microbes are mass-spectrometrically measured and compared after being cultured in media with and without added antibiotics, for example. Here the microbes are cultured in centrifuge tubes with and without antibiotics, for example. After culturing, the microbes are centrifuged out, rinsed, and then disrupted with acids and acetonitrile in the centrifuge tubes so that their soluble proteins are released. A small amount (around one microliter) of this liquid with disrupted microbial cells is prepared onto the sample support, dried and then coated with a small amount of matrix solution (also around one microliter). The dissolved proteins are embedded into the matrix crystals which are produced in the drying process. The samples with matrix crystals and embedded proteins are bombarded with laser light pulses in the mass spectrometer, causing ions of the protein molecules to be formed in the vaporization plasma. Measuring their time-of-flight in a time-of-flight mass spectrometer produces the mass spectrum of the microbe, which essentially consists of the protein peaks.
The similarity between the two mass spectra allows only limited conclusions to be drawn about the resistance of the microbes. If susceptible microbes are only inhibited in their growth or killed, without being destroyed, as for example klebsiellae from the family of the enterobacteriaceae, the resulting mass spectrum is practically identical to that of the microbes from media without antibiotics. This is because the formation of the mass spectra which are obtained using ionization by matrix-assisted laser desorption (MALDI) is only slightly dependent on the quantity of microbes in the sample. Sample preparations with ten thousand microbes provide practically the same mass spectra as sample preparations with ten million microbes, which is ideal for an identification, but not for identifying the resistance. If merely the growth of the microbes is stopped, the same mass spectra result, because the only difference is in quantity, which does not show up in the mass spectra.
As is explained further in the Govorun patent, the resistance can also be identified by adding a second type of microbe to the microbes under investigation; this second type provides a very different mass spectrum, is resistant, and definitely continues to grow in the presence of the antibiotic. The mass spectrum with the superimposed proteins of both microbes should then show the differences in growth. Unfortunately, this method has proved to be not very practicable in routine work for various reasons; it assumes at least rough quantitative determinations of the microbes used, approximately equal growth rates in the culture medium used, and that the reference microbes are resistant to many antibiotics.
The microbes whose resistance is to be determined are preferably present in sufficient quantities in a sufficiently pure form. They can form colonies on an agar, or also exist as microbes from a blood culture, for example. With agar cultures, it is common practice to use microbes from not just one colony for the test, but to subject the microbes from at least five colonies together to this test in order to identify the possible presence of a resistant microbe among non-resistant microbes of the same species. A trained and experienced laboratory technician is generally able to recognize colonies of the same species of microbe and to harvest them. They must then be mixed and divided up for the cultures. Blood cultures naturally contain a mixture of the different microbes which were transmitted in the infection.
As a rule, a resistance determination (including one according to Govorun) is often preceded by the identification of the microbes which have grown on the agar culture or in the blood culture. It is helpful here, especially for the method according to Govorun, to know the microbe species and its growth rate. It is also usually the case that the antibiotics against which this microbe can be resistant are also known. Furthermore, the minimum inhibitory concentrations (MIC) for susceptible microbes are also usually known. This means that cultures with these antibiotics in suitable concentrations can be prepared; the minimum culture times for the Govorun method are given by the known growth rates.
In view of the foregoing, there is a need to provide a mass-spectrometric method with which the resistance of microbes to one or more antibiotics can be determined with certainty, at low cost, in a largely automated manner, and, most importantly, at high speed, in particular for fast-growing and thus especially dangerous pathogens in about an hour. It is preferable that the method can be carried out using the same routine mass spectrometers which are also used for the mass-spectrometric identification of the microbes.